CCN2 Increases TGF-β Receptor Type II Expression in Vascular Smooth Muscle Cells: Essential Role of CCN2 in the TGF-β Pathway Regulation

The cellular communication network factor 2 (CCN2/CTGF) has been traditionally described as a mediator of the fibrotic responses induced by other factors including the transforming growth factor β (TGF-β). However, several studies have defined a direct role of CCN2 acting as a growth factor inducing oxidative and proinflammatory responses. The presence of CCN2 and TGF-β together in the cellular context has been described as a requisite to induce a persistent fibrotic response, but the precise mechanisms implicated in this relation are not described yet. Considering the main role of TGF-β receptors (TβR) in the TGF-β pathway activation, our aim was to investigate the effects of CCN2 in the regulation of TβRI and TβRII levels in vascular smooth muscle cells (VSMCs). While no differences were observed in TβRI levels, an increase in TβRII expression at both gene and protein level were found 48 h after stimulation with the C-terminal fragment of CCN2 (CCN2(IV)). Cell pretreatment with a TβRI inhibitor did not modify TβRII increment induced by CCN2(VI), demonstrating a TGF-β-independent response. Secondly, CCN2(IV) rapidly activated the SMAD pathway in VSMCs, this being crucial in the upregulation of TβRII since the preincubation with an SMAD3 inhibitor prevented it. Similarly, pretreatment with the epidermal growth factor receptor (EGFR) inhibitor erlotinib abolished TβRII upregulation, indicating the participation of this receptor in the observed responses. Our findings suggest a direct role of CCN2 maintaining the TGF-β pathway activation by increasing TβRII expression in an EGFR-SMAD dependent manner activation.


Introduction
The transforming growth factor beta (TGF-β) belongs to the TGF-β growth factor superfamily implicated in cell division, differentiation, migration, adhesion, organization and death [1][2][3]. The relevant role of TGF-β in cellular homeostasis has been widely demonstrated and the deregulation of their related pathways has been associated with several human pathologies including cancer, autoimmune disorders and cardiovascular diseases [4,5]. The classical TGF-β pathway activation starts with TGF-β binding to its serintreonin kinase receptor type II (TβRII) which leads to receptor type I (TβRI) activation. The active TβRI, enlists and phosphorylates the receptor-regulated SMADs proteins (R-SMADs:

CCN2(IV) Increases TβRII Levels in Cultured VSMCs after 48 h
In order to evaluate whether CCN2 could modulate TGF-β receptors levels, culture VSMCs were treated with the recombinant CCN2(IV) at different doses (50 or 100 ng/mL). CCN2(IV) significantly increased TβRII protein levels in VSMCs at both doses compared with basal condition cells after 48 h ( Figure 1A). Conversely, the treatment with TGF-β (10 ng/mL) induced a significant decrease in the TβRII levels ( Figure 1A). On the other hand, neither CCN2(IV) nor TGF-β modified TβRI protein levels ( Figure 1B). Similarly, while CCN2(IV) also increased TβRII mRNA levels after 48 h at 50 and 100 ng/mL doses, TGF-β dramatically decrease them ( Figure 1C). Finally, CCN2(IV) treatment did not modify TβRI and TGF-β mRNA levels at any studied dose after 48 h ( Figure 1D,E). was to evaluate whether CCN2 modulate TGF-β receptors levels in VSMCs and to describe the potential mechanisms implicated.

CCN2(IV) Increases TβRII Levels in Cultured VSMCs after 48 Hours
In order to evaluate whether CCN2 could modulate TGF-β receptors levels, culture VSMCs were treated with the recombinant CCN2(IV) at different doses (50 or 100 ng/mL). CCN2(IV) significantly increased TβRII protein levels in VSMCs at both doses compared with basal condition cells after 48 h ( Figure 1A). Conversely, the treatment with TGF-β (10 ng/mL) induced a significant decrease in the TβRII levels ( Figure 1A). On the other hand, neither CCN2(IV) nor TGF-β modified TβRI protein levels ( Figure 1B). Similarly, while CCN2(IV) also increased TβRII mRNA levels after 48 h at 50 and 100 ng/mL doses, TGFβ dramatically decrease them ( Figure 1C). Finally, CCN2(IV) treatment did not modify TβRI and TGF-β mRNA levels at any studied dose after 48 h ( Figure 1D,E). CCN2(IV)-incubated vascular smooth muscle cells (VSMCs) showed increased protein levels of TβRII at doses of 50 ng/mL and 100 ng/mL after 48 h of treatment, whereas TGFβ (1 ng/mL) treatment decreased TβRII protein levels compared to non-treated VSMCs (A). Protein levels of TβRI were not modified neither by CCN2(IV) nor TGFβ (B). After 48 h of treatment, CCN2(IV) increased TβrII (C), but not TβrI (D) and Tgf-β (E) mRNA levels, whilst TGF-β stimulation decreased TβrII and did not modify TβrI and Tgf-β mRNA levels. Data are presented as mean ±SEM of 4 independent experiments. * p < 0.05 increased vs. Basal; † p < 0.05 decreased vs. Basal.

CCN2(IV) Activates the SMAD Pathway in Cultured and Aortic VSMCs at Short Times
To test whether CCN2 directly activate the SMAD pathway, culture VSMCs were incubated with CCN2(IV) at different times. After 10, 15 and 20 min, CCN2(IV) induced a significant SMAD pathway activation observed by increased levels of phosphorylated SMAD3 (Figure 2A

CCN2(IV) Activates the SMAD Pathway in Cultured and Aortic VSMCs at Short Times
To test whether CCN2 directly activate the SMAD pathway, culture VSMCs were incubated with CCN2(IV) at different times. After 10, 15 and 20 min, CCN2(IV) induced a significant SMAD pathway activation observed by increased levels of phosphorylated SMAD3 (Figure 2A, p-SMAD3) and SMAD2 ( Figure 2B, p-SMAD2). The SMAD pathway activation was confirmed by translocation of p-SMAD2 ( Figure 2C) and SMAD 4 ( Figure 2D) into the VSMCs nuclei after 10 and 20 min. As observed in vitro, the in vivo

CCN2(IV) Increases TβRII Expression in VSMCs by TGFβ -Independent SMAD Activation
The potential role of SMAD pathway activation induced by CCN2(IV) in the regulation of TβRII expression was evaluated by using the SMAD3 inhibitor SIS3. The protein levels evaluation demonstrated that preincubation of VSMCs with SIS3 for 1 h inhibited TβRII upregulation induced by CCN2(IV) after 48 h of treatment ( Figure 4A), demonstrating a direct role of SMAD activation in this process. On the other hand, preincubation of VSMCs for 1 h with galunisertib, a potent TβRI inhibitor, did not modulate TβRII levels in CCN2(IV) treated cells ( Figure 4B), indicating a TGF-β independent response.

CCN2(IV) Increases TβRII Expression in VSMCs by TGF-β -Independent SMAD Activation
The potential role of SMAD pathway activation induced by CCN2(IV) in the regulation of TβRII expression was evaluated by using the SMAD3 inhibitor SIS3. The protein levels evaluation demonstrated that preincubation of VSMCs with SIS3 for 1 h inhibited TβRII upregulation induced by CCN2(IV) after 48 h of treatment ( Figure 4A), demonstrating a direct role of SMAD activation in this process. On the other hand, preincubation of VSMCs for 1 h with galunisertib, a potent TβRI inhibitor, did not modulate TβRII levels in CCN2(IV) treated cells ( Figure 4B), indicating a TGF-β independent response.

CCN2(IV) Increases TβRII Expression in VSMCs by TGF-β -Independent SMAD Activation
The potential role of SMAD pathway activation induced by CCN2(IV) in the regulation of TβRII expression was evaluated by using the SMAD3 inhibitor SIS3. The protein levels evaluation demonstrated that preincubation of VSMCs with SIS3 for 1 h inhibited TβRII upregulation induced by CCN2(IV) after 48 h of treatment ( Figure 4A), demonstrating a direct role of SMAD activation in this process. On the other hand, preincubation of VSMCs for 1 h with galunisertib, a potent TβRI inhibitor, did not modulate TβRII levels in CCN2(IV) treated cells ( Figure 4B), indicating a TGF-β independent response.

TβRII Expression Induced by CCN2(IV) in VSMCs Is Mediated by the EGF Receptor
Considering our previous published results describing the ability of CCN2 to directly bind to and activate the epidermal growth factor receptor (EGFR) [28,50], the next aim was to determine the participation of this receptor in the observed results. The preincubation of VSMCs with the EGFR inhibitor erlotinib 1-h prior CCN2(IV) administration, prevented TβRII upregulation after 48 h ( Figure 4C).

Discussion
The present study points out a direct role of CCN2 increasing TβRII levels and, therefore, suggests its participation exerting positive feedback in the TGF-β pathway activation. On the contrary, TGF-β induces a reduction in TβRII levels, indicating the complexity of this pathway regulation. Our results provide a potential explanation of the previously described relevance of CCN2 maintaining the TGF-β profibrotic response and open new ways to future studies in this field.
The regulation of the TGF-β pathway comprises a wide range of components and factors, which lead into the activation of a large list of genes [51]. Among the latter, CCN2 plays an essential role in the profibrotic response induced by TGF-β [52,53]. TGF-β pathway components levels have been described to be essential in the regulation of the TGF-β activation [48]. Upon their activation, the heteromeric TβRI/TβRII complexes are rapidly internalized into the cytoplasm by, at least, two different processes: the classical clathrin-dependent pathway, which helps SMAD activation, and a lipid raft-caveolin dependent process which mediates the receptor degradation [54]. Consequently, the specificity of signaling pathway activation and the biological effects of TGF-β are modulated by TGF-β receptor levels [47]. At vascular level, the relevance of TGF-β receptors has been described in several pathologies. Thus, the cell-phenotype conversion from an antiproliferative to a profibrotic response after TGF-β stimulation observed in VSMCs derived from human atherosclerotic and restenotic lesions, was attributed to the decreased ratio of TβRII/TβRI [55][56][57]. Interestingly, one of the most used drugs for atherosclerosis treatment, statins, have demonstrated to increase TβRII expression, as well as CCN2 production in cultured VSMCs [58]. Consequently, atorvastatin treatment increased TβRII expression in the atheroma plaque in an experimental model of atherosclerosis in Apolipoprotein E Knockout mice, which was associated to beneficial effects, including amelioration of disease progression and stabilization of the atheroma plaque by increased CCN2 expression and collagen content [58]. These data suggest an interrelation between CCN2 and TβRII regulation both in vitro and in vivo in VSMCs. Here we demonstrate that CCN2 increased TβRII expression in cultured VSMCs at both protein and gene level after 48 h, while TβRI levels remained unaltered. Contrarily, TGF-β decreased TβRII levels after 48 h of stimulation, which could correspond to the above-mentioned TGF-β receptor degradation. These findings suggest a direct role of CCN2 maintaining positive feedback in the TGF-β response.
CCN2 exerts a dual role in the vasculature not only acting as a growth factor, but also maintaining vascular homeostasis [22,59,60]. This feature could explain the different results obtained modulating CCN2 levels in experimental cardiovascular pathologies showing the benefits of both blocking [24,61,62] or overexpressing CCN2 [37,38,40,41,63], depending on the disease. Regarding gene expression regulation, CCN2 knockout mice die shortly after birth by respiratory failure due to its essential role in coordinating chondrogenesis and angiogenesis during skeletal development [64]. In adult mice, tamoxifen-dependent CCN2 deletion ameliorated renal fibrosis [65], but it did not improve cardiac fibrosis and hypertrophy [66]. Recently, our group has demonstrated the relevance of CCN2 on maintaining vascular wall homeostasis in a model of Ang II-induced vascular damage. In this sense, acquired CCN2 deletion in adult mice predispose to rapid aortic aneurysms development and rupture after Ang II administration [67]. These results are similar to those observed in experimental mice models combining TGF-β neutralization with Ang II infusion, which enhanced AngII-induced aortic rupture and aneurysm in both thoracic and abdominal regions [68,69]. Although further studies are necessary to further evaluate this hypothesis, the present results open new potential mechanisms in which CCN2, by increasing TβRII expression in VSMCs, could exert positive feedback in the TGF-β pathway activation, contributing to the TGF-β-beneficial effects described in some vasculopathy situations [70]. Regarding CCN2-growth factor actions, our previous studies described the ability of CCN2 to induce pro-oxidative and pro-inflammatory responses in cultured VSMCs and mice aorta [28]. In the present study we demonstrated that CCN2 also activates SMAD pathway in cultured VSMCs at early time-points leading to TβRII production after 48 h. We have previously described that CCN2 directly binds to and activates EGFR [28,50] to induce pro-oxidative and pro-inflammatory responses in VSMCs. In this new study we further strengthen the EGFR participation in CCN2 responses, demonstrating that this receptor is essential to induce TβRII overexpression mediated by CCN2(IV) in VSMCs. Altogether our data suggest a potential mechanism implicated in TβRII overexpression induced by CCN2(IV) that include EGFR and SMAD pathway activation ( Figure 5).
thoracic and abdominal regions [68,69]. Although further studies are necessary to further evaluate this hypothesis, the present results open new potential mechanisms in which CCN2, by increasing TβRII expression in VSMCs, could exert positive feedback in the TGF-β pathway activation, contributing to the TGF-β-beneficial effects described in some vasculopathy situations [70]. Regarding CCN2-growth factor actions, our previous studies described the ability of CCN2 to induce pro-oxidative and pro-inflammatory responses in cultured VSMCs and mice aorta [28]. In the present study we demonstrated that CCN2 also activates SMAD pathway in cultured VSMCs at early time-points leading to TβRII production after 48 h. We have previously described that CCN2 directly binds to and activates EGFR [28,50] to induce pro-oxidative and pro-inflammatory responses in VSMCs. In this new study we further strengthen the EGFR participation in CCN2 responses, demonstrating that this receptor is essential to induce TβRII overexpression mediated by CCN2(IV) in VSMCs. Altogether our data suggest a potential mechanism implicated in TβRII overexpression induced by CCN2(IV) that include EGFR and SMAD pathway activation ( Figure 5).

Figure 5.
Graphical scheme of proposed mechanism by which CCN2 may be triggering the expression of TβRII, compared to canonical TGF-β pathway activation.

Experimental Mice Model
Experimental animal studies were performed in adult male C57BL/6 mice (9-12 weeks old, 20 g; Harlan Interfauna lbérica, S.A., Barcelona, Spain) and maintained in the animal facilities of the "Instituto de Investigación Sanitaria Fundación Jiménez Díaz" (IIS-FJD) fed with standard diet and water ad libitum, under special pathogen-free conditions and normal light-dark cycles. All the procedures with animals were performed according to the European Community (RD53/2013) and IIS-FJD Animal Research Ethical Committee guidelines (PROEX 065/18). CCN2(IV) administration was performed as previously described [28]. Briefly, mice were intraperitoneally injected with CCN2(IV) (2.5 ng/g of body weight, dissolved in saline) and were euthanatized after 24 h under anesthesia (Isofluorane; Abbott Laboratories, Madrid, Spain). Aortas were collected, dissected free of fat and connective tissue, fixed in paraformaldehyde and embedded in paraffin. A control

Experimental Mice Model
Experimental animal studies were performed in adult male C57BL/6 mice (9-12 weeks old, 20 g; Harlan Interfauna lbérica, S.A., Barcelona, Spain) and maintained in the animal facilities of the "Instituto de Investigación Sanitaria Fundación Jiménez Díaz" (IIS-FJD) fed with standard diet and water ad libitum, under special pathogen-free conditions and normal light-dark cycles. All the procedures with animals were performed according to the European Community (RD53/2013) and IIS-FJD Animal Research Ethical Committee guidelines (PROEX 065/18). CCN2(IV) administration was performed as previously described [28]. Briefly, mice were intraperitoneally injected with CCN2(IV) (2.5 ng/g of body weight, dissolved in saline) and were euthanatized after 24 h under anesthesia (Isofluorane; Abbott Laboratories, Madrid, Spain). Aortas were collected, dissected free of fat and connective tissue, fixed in paraformaldehyde and embedded in paraffin. A control saline-injected group was also studied (n = 7 mice per group). The purity of CCN2(IV) (endotoxin levels < 0.01) was evaluated by MALDI-TOF (data not shown).

Cell Cultures
Vascular smooth muscle cells (VSMCs) came from mice aorta cell line MOVAS (ATCC CRL-2797; Barcelona, Spain). VSMCs were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2% L-glutamine 200 mM, 100 U/mL penicillin, 100 U/mL streptomycin and 0.2 mg/mL G-418 (all reagents were obtained from Sigma Chemical, MO, USA). Every experiment was performed at 80% of confluence, as well as in growth-arrested cells conditioned by serum starvation during the 24 h prior to stimuli. Cells were treated with recombinant C-terminal CCN2 (CCN2(IV)) (Peprotech, London, UK) as stimulus at concentrations of 50 and 100 ng/mL evaluated at different time-points. According to previous studies of our group [58,71], SMAD pathway activation was evaluated at early time-points (5 to 20 min) whereas TGF-β receptors levels were assessed after 48 h of CCN2(IV) treatment. The following pharmacological inhibitors were used to study different pathways: SIS3 (Selleck Chemicals, Berlin, Germany), as a SMAD3 phosphorylation inhibitor [72], galunisertib (Selleck Chemicals) a TβRI antagonist [73], and erlotinib, an EGFR inhibitor (Vichem Chemie Research, Budapest, Hungary).

qPCR Analysis
Total mRNA was obtained by using TRIzol method as previously described (Invitrogen) and retro-transcribed into cDNA by using the Reverse Transcription kit (Applied biosystems, Life Technologies, Inchinnan, UK). Multiplex real time-PCR was performed using fluorogenic primers designed by the Assay-on-Demand mouse gene expression products (Applied Biosystems): Tgfb (Mm01178820_m1), Tgfbr1 (Mm00436964_m1) and Tgfbr2 (Mm03024091_m1) (FAM). Glyceraldehyde 3-phosphate dehydrogenase (Gapdh) (Mm99999915_g1, VIC) was used as endogen control to normalized data. The mRNA copy number were calculated for each sample by the instrument software (ABIPrism 7500 Fast sequence detection PCR system software (Applied Biosystems)) using Ct value ("arithmetic fit point analysis for the lightcycler"), and results were expressed in n-fold calculated vs. control.

Statistical Analysis
Data are expressed as mean ± standard error of the mean (SEM). Normality distribution was tested by using Shapiro-Wilk test. If the samples followed a Gaussian distribution or not, means were compared by t-student or Mann-Whitney statistical test respectively. Every statistical analysis was conducted using GraphPad Prism 8.0 (GraphPad Software, San Diego, CA, USA). Values of p < 0.05 were considered statistically significant.

Conclusions
The present study contributes to extend the complex mechanism implicated in the TGF-β pathway regulation suggesting that CCN2 expression induced by TGF-β positively regulates TβRII synthesis, which could compensate TβRII degradation induced by TGFβ and, therefore, explain the essential role of CCN2 maintaining the TGF-β-mediated profibrotic responses.

Conflicts of Interest:
The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. The results presented in this paper have not been published previously in whole or part.